This invention relates to inhibitors of the amino peptidase activity of dipeptidyl peptidase type IV (DP-IV).
DP-IV is a postproline cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of polypeptides. DP-IV will also remove Xaa-Ala dipeptides from amino termini, albeit less efficiently. DP-IV is present in many mammalian cells and tissues, for example, renal tubule cells, intestinal epithelium, and blood plasma. It is also present on the surface of CD4+ and some CD8+ T-cells. It is thought to be involved in the regulation of the immune response; occurrence of DP-IV on a cell surface is associated with the ability of cells to produce interleukin-2 (IL-2). DP-IV is also referred to as dipeptidyl-peptide hydrolase DAP-IV or DPP-IV; it is assigned EC number 3.4.14.5.
Three different inhibitors of DP-IV are known. One of these is a suicide inhibitor: N-Ala-Pro-O-(nitrobenzyl-) hydroxylamine. (The standard three letter amino acid codes are used in this application; O represents oxygen.) Another is a competitive inhibitor: e-(4-nitro) benzoxycarbonyl-Lys-Pro. The third is a polyclonal rabbit anti-porcine kidney DP-IV immunoglobulin.
The enzymatic activity of DP-IV involves cleaving of a dipeptide from the free amino terminus of a polypeptide. DP-IV has a preference for cleaving after a proline, i.e., a proline in the penultimate position from the amino terminus. A free amino terminus is required; thus, DP-IV is a postproline cleaving enzyme with a specificity for removing an N-terminal Xaa-Pro dipeptide from a polypeptide (where Xaa can be any amino acid, including proline). DP-IV also will remove a Xaaxe2x80x2-Ala dipeptide from an amino terminus of a polypeptide when Xaaxe2x80x2 is an amino acid with a bulky side group, e.g., tyrosine.
This invention concerns provision of potent inhibitors of the enzymatic activity of DP-IV. Generally, an xcex1-amino boronic acid analog of proline (boroPro is used to designate one such analog which has the carboxyl group of proline replaced with a B(OH)2 group, where (OH)2 represents two hydroxyl groups and B represents boron) is bonded to an amino acid to form a dipeptide with boroPro as the carboxy terminal residue. These dipeptide prolyl-boronic acids are potent and highly specific inhibitors of DP-IV, with Ki values in the nanomolar range.
Dipeptides having the boroPro moiety are relatively unstable; thus, we have designed inhibitors having at least two other amino acid residues. Generally, the structure of these inhibitors is X-Pro-Y-boroPro where X and Y are chosen from any amino acid residue (including proline). This tetrapeptide may be lengthened at its amino-terminus by addition of one or more dipeptides, each dipeptide having the general formula Z-Pro or Z-ala, where each Z independently is any amino acid residue (including proline). This general structure is defined in more detail below. These inhibitors function as inhibitors of DP-IV because each dipeptide portion is a substrate for DP-IV and the final product of the reaction of such an inhibitor with DP-IV is the dipeptide inhibitor Y-boroPro. The amino terminus of these inhibitors must not be blocked or they lose their inhibitory capacity for DP-IV, since DP-IV cannot cleave a dipeptide from a blocked N-terminal polypeptide.
Thus, in a first aspect, the invention features an inhibitory compound having the structure: Group I-Group II. Group I has the structure: 
where H represents a hydrogen; C represents a carbon; O represents an oxygen; N represents a nitrogen; each R, independently, is chosen from the group consisting of the R groups of an amino acid, including proline; each broken line, independently, represents a bond to an H or a bond to one R group, and each Hxe2x80x2 represents that bond or a hydrogen; and p is an integer between 0 and 4 inclusive.
Alternatively, Group I has the structure: 
where n is between 0 and 3 inclusive, each G2 and G3 independently is H or C1-3 (one to three carbon atoms) alkyl, G1 is NH3 (H3 represents three hydrogens), 
(H2 represents two hydrogens), or NG4, where G4 is 
where G5 and G6 can be NH, H, or C1-3 alkyl or alkenyl with one or more carbons substituted with a nitrogen. G1 bears a charged, and G1 and Group II do not form a covalently bonded ring structure at pH 7.0. Group I may also have the structure: 
where one or two of the a, b, c, d, e, and f group is N, and the rest are C, and each S1-S6 independently is H or C1-C3 alkyl. Group I may also include a five membered unsaturated ring having two nitrogen atoms, e.g., an imidazole ring. Group II has the structure: 
where T is a group of the formula: 
where each D1 and D2, independently, is a hydroxyl group or a group which is capable of being hydrolysed to a hydroxyl group in aqueous solution at physiological pH; a group of the formula: 
where G is either H, fluorine (F) or an alkyl group containing 1 to 20 carbon atoms and optional heteroatoms which can be N, S (sulfur), or O; or a phosphonate group of the formula; 
where each J, independently, is O-alkyl, N-alkyl, or alkyl. Each O-alkyl, N-alkyl or alkyl includes 1-20 carbon atoms and, optionally, heteroatoms which can be N, S, or O. T is generally able to form a complex with the catalytic site of a DP-IV. 
and each R1, R2, R3, R4, R5, R6, R7, and R8, separately is a group which does not significantly interfere with site specific recognition of the inhibitory compound by DP-IV, and allows a complex to be formed with DP-IV.
In preferred embodiments, T is a boronate group, a phosphonate group or a trifluoroalkyl ketone group; each R1-R8 is H; each R1 and R2 is H, and each Y is the CH2-CH2; each R is independently chosen from the R group of proline and alanine; the inhibitory compound has a binding or dissociation constant to DP-IV of at least 10xe2x88x929M, 10xe2x88x928M or even 10xe2x88x927M; the inhibitory compound is admixed with a pharmaceutically acceptable carrier substance; and each D1 and D2 is, independently, F, or D1 and D2 together are a ring containing 1 to 20 carbon atoms, and optionally heteroatoms which can be N, S, or O.
In a second aspect, the invention features a method for inhibiting the enzymatic activity of DP-IV in a mammal. The method includes administering to the mammal an effective amount of an inhibitory compound described above. Most preferably, the amount of compound administered is between 1 and 500 mg/kilogram of animal treated/day.
In a third aspect, the invention features an inhibitor of DP-IV, having the structure: 
wherein m is an integer between 0 and 10, inclusive; A and Axe2x80x2 are L-amino acid residues (for glycine there is no such distinction) such that the A in each repeating bracketed unit can be a different amino acid residue; the C bonded to B is in the L-configuration; the bonds between A and X, Axe2x80x2 and C, and between Axe2x80x2 and N are peptide bonds; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolysed to a hydroxyl group at physiological pH. By xe2x80x9cthe C bonded to B is in the L-configurationxe2x80x9d is meant that the absolute configuration of the C is like that of an L-amino acid.
Thus the 
group has the same relationship to the C as the xe2x80x94COOH group of an L-amino acid has to its xcex1 carbon. In various preferred embodiments, A and Axe2x80x2 are independently proline or alanine residues; m is 0; X1 and X2 are hydroxyl groups; the inhibitor it L-Ala-L-boroPro; and the inhibitor is L-Pro-L-boroPro.
In a fourth aspect, the invention features a method for inhibiting DP-IV in a mammal. The method includes administering to the mammal an effective amount of the compound: 
described above. In a preferred embodiment, the amount is 1 mg/kg of the mammal per day to 500 mg/kg of the mammal per day.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.